In vitro models for non-alcoholic fatty liver disease: Emerging platforms and their applications

Summary Non-alcoholic fatty liver disease (NAFLD) represents a global healthcare challenge, affecting 1 in 4 adults, and death rates are predicted to rise inexorably. The progressive form of NAFLD, non-alcoholic steatohepatitis (NASH), can lead to fibrosis, cirrhosis, and hepatocellular carcinoma. However, no medical treatments are licensed for NAFLD-NASH. Identifying efficacious therapies has been hindered by the complexity of disease pathogenesis, a paucity of predictive preclinical models and inadequate validation of pharmacological targets in humans. The development of clinically relevant in vitro models of the disease will pave the way to overcome these challenges. Currently, the combined application of emerging technologies (e.g., organ-on-a-chip/microphysiological systems) and control engineering approaches promises to unravel NAFLD biology and deliver tractable treatment candidates. In this review, we will describe advances in preclinical models for NAFLD-NASH, the recent introduction of novel technologies in this space, and their importance for drug discovery endeavors in the future.


INTRODUCTION
Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease, mainly affecting patients with obesity and type 2 diabetes mellitus (T2DM). NAFLD is the umbrella-term used for a range of conditions caused by a build-up of fat in the liver and encompasses isolated fatty liver (steatosis) and non-alcoholic steatohepatitis (NASH) with increasing degrees of liver inflammation, fibrosis (scarring), and potentially cirrhosis (end-stage fibrosis with vascular changes and functional impairment). Its worldwide prevalence is currently 25%, with 41% of the patients ultimately developing fibrosis (Younossi et al., 2016) and is projected to increase further by up to 18.3% in some developed countries by 2030 (Estes et al., 2018). Sedentary lifestyles and booming economic growth in the most developed countries sustain such trends, which see NASH becoming the leading cause of liver transplant (Zou et al., 2020).
To date, there are no licensed pharmacological treatments for NASH. While bariatric surgery is effective in a small proportion of patients , diet and exercise remain the cornerstone of disease management, due to evidence that a 10% body weight loss allows a reversion of steatosis, inflammation, and even fibrosis (Vilar-Gomez et al., 2015). However, the difficulty of achieving and sustaining this supports the need for disease-specific pharmacological treatment. Current medical treatment also comprises drugs for comorbidities related to NAFLD. Patients with or without T2DM may be treated with anti-diabetic medications. A recent systematic review of 18 trials (Blazina and Selph, 2019) presented evidence that supports the efficacy of some diabetes drugs in reducing liver fat and resolving NASH (e.g., pioglitazone, liraglutide), although associated weight gain with pioglitazone warrants caution. Blazina and Selph (2019) concluded that larger trials are needed to elucidate the benefits and risks of diabetes pharmacotherapy in NAFLD patients.
Given the complexity of NAFLD pathophysiology and the phenotypic heterogeneity, both genetic and environmental factors are thought to contribute to disease progression and susceptibility (Eslam et al., 2018). Several genes that participate in lipid droplet remodeling, hepatic very low density (VLDL) secretion and generation of predominant molecular species of phosphatidylinositol (PI) in cell membranes have been identified as NAFLD genetic variants. Specifically, the most well-described variants are patatin-like phospholipase domain-containing protein 3 (PNPLA3) I148M (Romeo et al., 2008), transmembrane 6 superfamily According to their origin, cell lines are either tumor-derived (commonly known as immortalized), primary cell lines, or pluripotent stem cells (PSC). Immortalized cell lines used for NAFLD research (e.g., HepaRG, THP-1 or LX-2), offering unlimited growth and stable phenotype, streamline standardized culture protocols and assay reproducibility (Ramboer et al., 2014). Nonetheless, mutations in immortalized cell lines can hamper translation of observations to the human in vivo condition; for instance, HepG2 is characterized by the PNPLA3 I148M mutation, which is known to affect some metabolic functions (Gunn et al., 2017). Primary human cells, isolated from liver tissue as hepatocytes or non-parenchymal cells (NPC), better resemble the in vivo phenotype (Wilkening et al., 2003). Indeed, harvesting of these cells from NAFLD patients enhanced the accuracy of drug metabolism studies (Ganji et al., 2015;Schwartz et al., 2020). However, their limited culture time and availability, especially for non-pathological (''healthy'') controls, as well as sample heterogeneity, are disadvantages (Zeilinger et al., 2016;Bell et al., 2018). When focusing on NAFLD modeling, inherent donor liver variability represents a double-edge sword, setting the basis for the investigation of genetic polymorphisms (Mü ller and Sturla, 2019). Lastly, PSC can be differentiated into liver-like cells from liver stem cells, induced pluripotent stem cells (iPSC) or embryonic pluripotent stem cells (ESC) (Hu and Li, 2015;Touboul et al., 2010;Takahashi et al., 2007). Cell differentiation, achieved via addition of growth factors and nutrients to the culture medium, results in phenotypic similarity to primary cell lines in addition to indefinite expansion in culture (Soret et al., 2021). Moreover, iPSC are the preferred PSC for investigation, the use of ESC being associated with ethical concerns (Zeilinger et al., 2016). Recent in vitro models with human PSC have been developed by Sinton et al. (2021); Holmgren et al. (2020); Coll et al. (2018) for NAFLD modeling. In Table 1, advantages and disadvantages of various cell lines used in NAFLD in vitro modeling are detailed.
Cell culture models 2D and 3D cultures have been extensively used in NAFLD research and in vitro modeling ( Figure 1, Table  S1), albeit to a different extent. We have performed a systematic review to observe the evolution and focus of in vitro models in NAFLD ( Figure S1). Our systematic review indicated that researchers predominantly favored 2D monocultures (59.4%) to more complex models (2D co-cultures (14%), spheroids (9.7%), organoids (7.3%), liver-on-a-chip (7.8%), collagen gel sandwiches (1.2%), and micropatterned cultures (0.6%). When analyzing the selection of cell culture systems over time ( Figure S2), we observed an increasing trend in the publication of 3D in vitro models, specifically in on-chip cultures. This suggests 3D culture systems are becoming more relevant in this field. Focusing on the large number of publications in monocultures in 2020, we note that these systems have been used in conjunction with additional in vivo ll OPEN  experiments, or as a benchmark for more sophisticated in vitro models. As an example, the insert in Figure S2 distinguishes publications in 2020 that have used monocultures as the only in vitro experiment from those that used them in combination with other cell culture models. We speculate that technological advances, standardizing the development of more complex and affordable solutions, might underlie this observation.
As reported above, 2D monoculture is the most established NAFLD in vitro model. In this system, steatosis induction is routinely performed by adding a mixture of unsaturated and saturated FFA to the culture medium. Typically, oleic acid (OA) and palmitic acid (PA) are selected, being the most abundant in humans (Hodson et al., 2008). According to our systematic search the most commonly used ratio for these FFA is 1:2 (PA:OA). This FFA ratio exposure emulates dietary conditions that are commonly observed in vivo, inducing hallmarks of NAFLD-NASH (e.g., cytoplasmic accumulation of TG in hepatocytes, ER stress, inflammation, and cell death) (Gomez-Lechon et al., 2007).
The low cost, ease of use, and suitability for high-throughput screening make 2D monocultures attractive for disease modeling. Here, the choice of cell sources constrains the NAFLD mechanistic studies enabled by the in vitro model. For example, Lyall et al. (2018) showed that hepatocyte-like cells derived from human ESC retained the ability to form the cytosine modification 5-hydroxy-methylcytosine (5hmC), rapidly lost when culturing HepG2, providing a means to elucidate the role of epigenetic dysregulation in NAFLD pathogenesis. Similarly, an hPSC-derived model was used to study the re-wiring of mitochondrial metabolism in early steatosis (Sinton et al., 2021). Monoculture models for NAFLD have also been valuable for investigating metabolic and fibrotic pathways (Graffmann et al., 2016;Kanda et al., 2018;Zhang et al., 2016), evaluating therapeutic compounds (Boeckmans et al., 2020;Armstrong et al., 2016;Anfuso et al., 2020), and assessing hepatic drug metabolism/toxicity (Gomez-Lechon et al., 2003).
Notwithstanding the advantages outlined above, 2D monocultures are unable to faithfully mimic in vivo NAFLD conditions due to the lack of interaction with NPC and disturbances in the extracellular environment. An improvement over this system is provided by 2D co-cultures, typically involving two cell types, such as hepatocytes and NPC, growing in a monolayer. As with monocultures, the steatotic phenotype can be induced upon treatment with FFA. Interestingly, a co-culture of Huh7 and LX-2 enabled Barbero-Becerra et al. (2015) to demonstrate the key role of hepatocyte-HSC crosstalk in the activation of HSC, a key feature of NASH. Co-cultures including macrophages (Zhong et al., 2017;Chen and Ma, 2019) have also elucidated the role of multiple inflammatory pathways in NAFLD and NASH. 2D cultures (e.g., monocultures and co-culture) are denoted by a single or several cell types growing in a monolayer. 3D cultures (e.g., collagen gel sandwich, spheroids, organoids or liver-on-a-chip) have been recently introduced to elucidate NAFLD, and their development can rely on bio-printing technology. Self-aggregated spheroids present an accumulation of carbon dioxide (CO 2 ) and waste in their core. Cell culture models are listed by increasing cost, longevity and complexity. While complexity correlates with ability of the model to accurately capture the disease phenotype, simpler models are better suited to high-throughput applications. iScience Review NAFLD and NASH can be induced in 2D co-cultures of PHH and iPSC (Parafati et al., 2018), yet their capacity as accurate in vitro models decays with time as they lose their xenobiotic metabolism and hormone response (Gurevich et al., 2020). 3D cultures can enhance these features, allow cell-cell interactions and cell polarity (Treyer and Mü sch, 2013;Godoy et al., 2013). They can provide new insights into complex physiological mechanisms and potential therapeutic targets, increasing our knowledge of NAFLD. Furthermore, they represent a suitable alternative to in vivo models at early stages of drug development (Romualdo et al., 2021;Beisner et al., 2021;Eilenberger et al., 2020). The main 3D in vitro models used for NAFLD -spheroids, organoids, and microfluidic devices-are detailed in the following section.
We classified the NAFLD in vitro models identified in our systematic search, according to their intended application, into NAFLD biology studies and compound testing. 47.9% of the publications relied on in vitro models to elucidate the role of different proteins, pathways or hallmarks of the disease; 18.8% of the papers detailed the development of new in vitro models to better mimic NAFLD biology and its progression; while 33.3% of the studies assessed the therapeutic potential of putative compounds and drug targets for NAFLD and NASH.
Despite a larger number of studies focused on the biology of NAFLD, the last 3 years have seen an increase in publications reporting compound testing ( Figure 2A). This, together with the simultaneous decline in the study of disease mechanisms in NAFLD, suggests a possible shift in research on the condition. Specifically, recent publications leverage available knowledge on NAFLD pathogenesis to devise strategies for treatment. We explored whether this shift in the field was promoted by a greater involvement of pharma, but did not observe any direct link, as most of the investigations remain hosted in academic settings. We also noted that despite the divergence in their severity and risk of progression, comparable effort has been devoted to NAFLD (51.4% of the studies) and NASH (43.7%). Only 4.9% of the investigations proposed in vitro models to mimic both conditions. Considering the varied clinical outcomes within the disease spectrum, it would be preferable to consider both NAFLD and NASH when investigating the disease. This is of paramount importance for compound testing since the mechanism of action (and potential efficacy) of drugs will depend on the disease stage.
In Figure 2B the distribution of the hallmarks of NAFLD/NASH across the studies is reported. Most publications concentrated on steatosis, followed by inflammation and fibrosis. The focus on steatosis is reflected Despite the majority published studies using in vitro models focused on NAFLD biology (orange), the past years have seen a marked increase in scientific articles presenting novel in vitro models (purple) or using available culture systems for compound testing (cyan). The concurrent decrease in investigations of the role of specific pathways and/or proteins in NAFLD pathogenesis suggests a shift in focus and efforts within the scientific community.
(B) Barplot showing the percentage of in vitro models that have been used to study steatosis, inflammation, fibrosis, or hepatocellular ballooning. For each disease hallmark, publications are clustered, according to their focus, in NAFLD (cyan), NASH (blue) or both NAFLD and NASH (dark blue). Steatosis is the most studied feature in NAFLD and NASH, followed by inflammation and fibrosis. Hepatocellular ballooning is the least studied feature in the scientific literature. For more information regarding the historical perspective of the evolution of in vitro models for NAFLD. See also Figure S2.
by the predominant use of monocultures of hepatocytes. Interestingly, hepatocellular ballooning has barely been explored in these studies. Although it is difficult to objectively measure the presence of ballooned hepatocytes in patients' biopsies (Kakisaka et al., 2018), they are a pathognomonic histological feature of NASH that should be considered when studying this disease.

3D TISSUE CULTURE MODELS IN NAFLD
Three-dimensional cultures are emerging as a bridge between the easy-to-use 2D cell cultures and more complex in vivo models. By maintaining hepatic cell types within a controlled microenvironment, 3D liver models better mimic the in vivo organ phenotype. From spheroids composed of a single cell type to multicellular aggregates including NPC and immune cells, 3D cultures can model the liver organotypic structure, retaining paracrine cell-cell interactions and metabolic functions for several weeks. Moreover, these models can be used to study fluid flow and perfusion when cultured on a chip. In this section, we will review the 3D culture models most recently proposed to recapitulate NAFLD. Specifically, we will detail the latest developments in spheroids, organoids, liver-on-a-chip and bio-printed platforms.
The collagen gel sandwich has been extensively used to culture liver cells (Engl et al., 2004;Sandwich, 2006;Kono et al., 1997;Choi and Choi, 2013;Wang et al., 2004). In this model, hepatocytes are seeded between two layers of collagen gel. The obtained structure better resembles the in vivo conditions, as demonstrated by reorganization of the cytoskeleton, improved morphology and polarity, and enhanced expression of liver-specific functions (Berthiaume et al., 1996). Within this model, hepatocytes maintained secretion of hepatic compounds for at least six weeks; a three-fold increase in the secretion window offered by monolayer collagen gel cultures (Dunn et al., 1991). Another collagen gel sandwich model, combining hepatocytes with liver sinusoidal endothelial cells (LSEC), mimicked the liver lobular architecture and showed stable secretion and metabolic activity for up to 4 weeks (Bale et al., 2015). While allowing long-term monitoring, the collagen gel sandwich has limitations. Indeed, when compared to 3D spheroids, a PHH-based collagen gel sandwich does not prevent hepatocyte dedifferentiation, as evidenced by perturbed glycolysis and gluconeogenesis, and shows lower sensitivity to long-term exposure to hepatotoxic compounds in cytotoxicity studies (Bell et al., 2018). Moreover, the collagen thickness may hinder cell-cell interactions (Soret et al., 2021). For these reasons, this model is seldom used to investigate NAFLD.
More recently, spheroids and organoids have become the 3D in vitro models of choice for studying NAFLD (Soret et al., 2021). Spheroids and organoids differ based on the origin and number of cell types in the aggregate, the culture environment or the complexity of their structure (Kang et al., 2021). In this review, we refer to spheroids as 3D structures produced by free-floating, spontaneous self-aggregation of cell lines in the presence or absence of an ECM that can recapitulate some functional aspects of the liver. In contrast, we denote organoids as 3D systems obtained by embedding tissue stem cells, progenitor cells or tissueresident cells isolated from liver specimens in a scaffold that imposes a liver-like spatial organization, thereby enabling modeling of the organotypic structure and function (Kang et al., 2021;Gunti et al., 2021). Figure 3 details how both NAFLD spheroids and organoids can be obtained from different sources.

Spheroid models
Since the first spheroid model in the 1970s (Sutherland et al., 1971), multiple methods have been developed to establish systems of increasing sophistication. In NAFLD modeling, the most popular method for spheroid culture is liquid overlay, where cell suspensions are cultured on ultra low-adhesive surface plates and formation of spheroids can be monitored in real-time (Gunti et al., 2021). Alternative methods for spheroid assembly have been extensively reviewed in the scientific literature (Kang et al., 2021;Gilazieva et al., 2020;Ryu et al., 2019).
The simplest hepatic spheroid model consists of a single cell type, usually HepG2 or PHH. It is worth noting that spheroids support culturing of differentiated PHH for a minimum of 3-4 weeks. Upon treatment with pathological concentrations of FFA, carbohydrates, cyclosporin A or insulin, hepatic spheroids enabled induction and investigation of steatosis for up to 5 weeks (Damelin et al., 2007;Bell et al., 2016;Kozyra et al., 2018). Specifically, these models have helped to elucidate the role of hepatocytes in NAFLD (Damelin et al., 2007;Chong et al., 2020), the mechanism by which IR elicits de novo lipogenesis and lipid accumulation (Kozyra et al., 2018), the identity of putative molecular targets or the pharmacological action of novel compounds to ameliorate the disease (Chong et al., 2020;Eilenberger et al., 2020;Bell et al., 2016 iScience Review of inter-individual variations as a determinant of susceptibility to steatosis development and progression (Dongiovanni et al., 2015). Thus, PHH-based spheroids could provide an ideal platform to demonstrate the impact of genetics on the variability in drug responses (efficacy and toxicity) among patients; the major cause of withdrawal of drugs from the market (Sim and Ingelman-Sundberg, 2011).
By co-culturing hepatocytes with NPC, beyond enhancing hepatocyte function in vitro, researchers can generate liver-like structures that recover the relevant mechanistic steps in NAFLD-NASH evolution (e.g. inflammation, HSC activation, and fibrogenesis). Hepatoma cell lines have been co-cultured with primary HSC to define pathways linked to liver steatosis and fibrosis (Salloum et al., 2021;Hurrell et al., 2020). Specifically, these co-culture systems showed enhanced collagen production, activation of HSC, upregulation of genes associated with fibrosis progression -such as collagen type I alpha I (COL1A1), TIMP metallopeptidase inhibitor 1 (TIMP1) or actin alpha 2 (ACTA2), and induction of pro-inflammatory cytokines. Bell et al. (2016) also co-cultured PHH-based spheroids with KC, HSC, and biliary cells, and highlighted that the coculture systems enable modeling of an inflammatory response, as measured by an increase in IL-6 secretion. Another example of a multicellular spheroid model has been proposed by Kachlishvili et al. (2020). The researchers combined a hepatocyte cell line with macrophages to study the effect of low-dose trans-resveratrol on lipid metabolism and pro-inflammatory processes. They showed a decrease in lipid accumulation due to enhanced poly(ADP-ribose) polymerase (PARP) activity, following treatment with the compound. As previous studies in monocultures hinted at an environment and cell-type dependent effect of resveratrol on metabolic pathways (Lan et al., 2017), the study by Kachlishvili et al. (2020) demonstrated the relevance of 3D spheroids for investigating the mechanism of action of drug candidates.
Spheroid models have further enhanced our understanding of genetic variants linked to NAFLD. In Tanaka   To induce the NAFLD phenotype, free fatty acids (FFA) supplementation is required. To form liver organoids, cells from liver specimens are extracted and isolated. Hence, they are exposed to selected growth factors in presence of a basement matrix. These liver organoids then differentiate into the specific cell lines and acquire the NAFLD phenotype upon FFA supplementation (light green panel). In addition, differentiated organoids can also be obtained from ESC/PSC or immortalized cell lines aggregated in liver spheroids. After dissociation of the aggregates and subsequent cultivation with a basement matrix, the differentiated organoids are ready to be used for NAFLD studies. The heterogeneity of the disease, its complexity, the self-organization of the cells or the life-span of the cultures is higher in organoids than in spheroids.

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iScience 25, 103549, January 21, 2022 11 iScience Review characterized by high fat accumulation and collagen production. This 3D model also captured antifibrotic effects following exposure to the compounds liraglutide and elafibranor. The same model was later selected as a screening platform for small molecule inhibitors, leading to the identification of momelotinib as a putative drug against the PNPLA3 I148M variant (Schwartz et al., 2020). PHH spheroids were additionally used to study the effect of the TM6SF2 E167K variant on steatosis establishment and progression in vitro . While the mutation led to higher intracellular lipids and upregulation of cholesterol biosynthesis and de novo lipogenesis, downregulation of genes implicated in NAFLD (e.g., ACADS, PNPLA2) revealed discrepancies with observed in vivo pathogenic mechanisms. However, the conclusions drawn from this study are limited by the small sample size exhibiting the TM6SF2 E167K variant (2 out of 5 PHH donors); a common challenge when working with PHH.
Despite their potential for disease modeling and drug efficacy testing, stem cell-based models are still mainly used in 2D cultures. Holmgren et al. (2020) assembled PSC-derived hepatocytes and HSC in spheroids, thereby avoiding the nuisance factor of spontaneous HSC activation induced by the stiffness of the ECM in 2D co-cultures (Wells, 2005). Gene expression analysis revealed similarities with PHH in glucose, lipid and cholesterol metabolism, reinforcing its suitability for NAFLD research. However, limited urea secretion and downregulation of enzymes involved in phase I and II drug metabolism hindered its use for drug screening studies.
Notwithstanding their simplicity and low cost, spheroid models offer limited control over their assembly and size when cultured in standard non-adhesive plates, presenting a scalability and reproducibility issue for liver spheres. Indeed, clusters of spheroids tend to merge into larger aggregates, challenging cell viability in culture by hampering both the intake of nutrients and oxygen diffusion (Underhill and Khetani, 2018a). To overcome this drawback, protocols detailing automated assembly of liver spheres have been published . In addition, novel approaches have incorporated the inclusion of hydrogel scaffolds, which direct the assembly of spatially isolated, controlled-size spheroids, in the development protocol (Ryu et al., 2019).

Organoid models
Liver organoids are 3D culture models obtained by embedding tissue stem cells, progenitor cells or tissue-resident cells isolated from liver specimens within a hydrogel ECM. Their development protocol allows tighter control over the spatial organization of the multicellular aggregate, better approximating the heterogeneity and organotypic structure observed in vivo (Gunti et al., 2021); once the exposure to growth factors and support matrices enabling differentiation of multiple cell types has been defined. Organoids supersede the ability of 2D cultures to model (patho)physiological processes, spanning cell differentiation, migration and cell-cell interactions. Furthermore, they retain genomic stability and, owing to wide expansion in culture, are suitable for long-term storage and high-throughput screening of drugs (Soret et al., 2021).
Murine-derived organoids have been widely used to model the NAFLD phenotype. After feeding wild-type or genetically modified mice with high-fat containing or specialized diets (such as the methionine and choline-deficient diet), researchers can harvest liver tissue for cell isolation and culture the cells within a hydrogel ECM stimulated with selected stem cell media or growth factors (Broutier et al., 2016). Recently, Elbadawy et al. (2020) modeled NAFLD progression via murine organoids assembled with hepatocytes and activated HSC isolated from animals at three distinct disease stages (mild, moderate and severe NASH) (Elbadawy et al., 2020). In doing so, Elbadawy et al. established a model to investigate the effect of genetic stability and lipid metabolism on NAFLD progression. The authors noticed larger size and enhanced release of pro-inflammatory cytokines in early stage organoid models, compared to later stage ones. Other murine organoids have been used to decipher liver metabolism and its response to therapeutic compounds. For example, Pittol et al. (2020)explored the dependency of metabolic functions on Farnesoid X receptor (FXR) isoforms; Sano et al. (2021) elucidated the associations among free amino acids, FFA and the Keap1-Nrf2 system -involved in the principal protective response to oxidative and electrophilic stresses (Turpaev, 2013) -in lipotoxic hepatocytes, while Liu et al. (2021) investigated the use of uridine to ameliorate NAFLD. Despite notable successes, murine-based organoids can only approximate the pathophysiology of human NAFLD (Kim et al., 2020), suggesting a human organoid model might provide a more suitable research platform.

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Several approaches for the development of human organoids to model NAFLD (and its extra-hepatic organ involvement) have recently been published. Ouchi et al. (2019) created a model from human PSC by codifferentiating epithelial and stromal lineages to form spheroids, later embedded in Matrigel and cultured with retinoic acid and maturation media. This protocol successfully yielded NPC including HSC-like cells, Kupffer-like cells and biliary stem cells; as well as hepatocyte-like and cholangiocyte-like cells, as confirmed by single-cell transcriptomics and FACS analysis. Upon FFA exposure, the organoids acquired a NAFLD phenotype denoted by steatosis, inflammation, hepatocyte ballooning and collagen production; hallmarks of NASH. Gurevich et al. (2020) opted instead for iPSC-derived cryopreservable hepatocytes sourced from NASH donors for organoid development. When co-cultured with physiologically-relevant ratios of isogenic HSC-like and Kupffer-like cells on collagen-coated ultra-low binding plates, the hepatocytes assembled in organoids amenable to observation for over 10 days. This system, characterized by the formation of bile canaliculi, proved useful for drug metabolism studies. In alignment with scientific evidence (Teo et al., 2014), efficient hepatocyte differentiation was promoted via glycogen synthase kinase 3 (GSK3) inhibition, which is known to induce endoderm differentiation. Similarly to Ouchi et al. (2019), treatment with FFA induced a dose-dependent accumulation of intracellular lipids and establishment of a NASH phenotype. McCarron et al. (2021) instead developed bipotent ductal organoids derived from end-stage NASH patient liver explants, with normal donor livers as controls. Strikingly, NASH patient-derived organoids were highly diverse in terms of metabolic and pro-inflammatory pathways and response to drugs. Moreover, the model was characterized by increased sensitivity to apoptosis, increased FFA induced lipid accumulation, and reduced albumin secretion-all typical features of advanced NASH. Another hepatic organoid model was developed using posterior foregut and hepatic endoderm. FFA induction elicited increasing lipid peroxidation and ROS. Interestingly, this 3D model highlighted disruption of the bile canalicular network with disease progression (Ramli et al., 2020). The organoid technology, especially when based on human stem cells, holds great promise for investigating NAFLD pathophysiology due to the ability to overcome confounding factors introduced by animal models. Thanks to enhanced stability and complexity, organoid models offer a viable platform for future drug development pipelines. The establishment of new cryopreservation (Gurevich et al., 2020) and reproducibility (Nuciforo and Heim, 2020) standards will certainly aid in unlocking the potential of organoids to offer a preclinical alternative to animal models in the near future. Indeed, validated organoids could provide an easy to image and more accurate model of human NAFLD. Recently, Takebe et al. (2017) targeted organoids reproducibility by establishing a technological platform to manufacture standardized liver buds starting from 3 committed progenitors on a large scale (>10 8 ).
We note that the bioengineering field is boosting efforts toward controlling organoids' cellular complexity, providing vascular networks, and enhancing organoid maturation (Garreta et al., 2020). For example, Guye et al. (2016) showcased the use of heterologous GATA-binding factor 6 (GATA6) expression to trigger codifferentiation events within an iPSC population and obtain, within 2 weeks, a complex tissue with liver-like phenotype. In Velazquez et al. (2021), engineering of gene regulatory networks in PSC-derived human liver organoids enabled advancement of maturity and vascularization of the obtained in vitro system. In addition, gene editing using CRISPR/Cas9 has also been used in bioengineering to alter and study the function of specific genes. For example, Abbey et al. (2020) deleted Tribbles-1 (TRIB1) -a gene associated with NAFLD, in human iPSC which were then used to create hepatic spheroids.

Liver-on-a-chip
Despite having proved informative for the study of NAFLD and complex diseases, a major drawback of the static 3D cultures is the lack of long-term control over fluid shear stress, nutrient/gas exchange or waste removal. To enable the automated control of such factors, microfluidic technology and liver-on-a-chip approaches have been adopted to model the liver microenvironment (Underhill and Khetani, 2018b iScience Review the cost and complexity of microfluidic setups constitutes a barrier to their diffusion in laboratories, the standardized fabrication of chips favors reproducibility, a hurdle in organoid research. Several liver-on-a-chip systems have recently been proposed to study NAFLD. The first appearing in the scientific literature approximated the liver architecture and microvasculature by culturing HepG2 cells in parallel microchannels mimicking the endothelial barrier (Gori et al., 2016). This microfluidic device demonstrated the ability of FFA perfusion to recapitulate steatosis. Since then, the combination of multiple cell types made on-chip cultures amenable to the study of cell-cell interactions, better reflecting the complexity of the hepatic microenvironment. Suurmond et al. (2019) co-cultured HUVEC with GelMa-encapsulated HepG2 to investigate NAFLD pathogenesis. Similarly to Gori et al., their system, distinguished by high cell viability and homogeneous cell distribution, enabled monitoring of steatosis establishment. Inclusion of KC in this in vitro model led to enhanced ROS expression and proinflammatory cytokine release. While HepG2 are a widely used cell line to investigate regulation of drug-metabolizing enzymes, PHH are the preferred choice in biotransformation studies, thanks to their in vivo-like functionality (Wilkening et al., 2003).
In light of their similarities to PHH, researchers increasingly based in vitro models on HepaRG cells. Suurmond et al. (2019) highlighted that by substituting HepG2 with HepaRG cells, their liver-on-a-chip displayed elevated levels of proinflammatory cytokines and improved reversibility of steatosis. HepaRG, in conjunction with KC, HUVEC and HSC encapsulated in GelMA, were used in the microfluidic model proposed by Cho et al. (2021). Through a 21-day life-span, the system recapitulated NASH features and showed neovascularization. Freag et al. (2021) also developed an on-chip model by co-culturing primary human hepatic cell lines (hepatocytes, KC, HSC and LSEC). In this system, lipotoxic conditions successfully generated the NASH phenotype, including hepatocellular ballooning. Furthermore, a PHH-based liver-ona-chip modeled the progressive intracellular lipid accumulation and reduction in cytochrome P activity observed in NAFLD patient samples (Kostrzewski et al., 2020). Interestingly, the presence of the PNPLA3 I148M variant in three of the donor samples allowed the authors to highlight that this variant can worsen the NASH phenotype through effects on HSC.
Despite the numerous advantages of liver-on-a-chip platforms to study NAFLD, the liver is not the only organ involved in the disease, with adipose tissue (Stojsavljevi c et al., 2014) and the gut (Scarpellini et al., 2014) known to play a key role in its progression. For this reason, it is important to study the interactions between multiple organs, and their contribution to disease establishment and progression. Multi-organ systems, also known as body-on-a-chip, are emerging as technological platforms to study the efficacy and safety of drugs (Picollet-D'hahan et al., 2021;Skardal et al., 2020). As most of the lipids accumulated in the liver are attributed to dietary intake, Lee and Sung (2018) focused on the gut-liver axis and developed an on-chip device generating hepatic steatosis. Their gut-liver-on-a-chip device consists of two chambers, incorporating Caco-2 (gut) and HepG2 (liver) cells, separated by a porous membrane and a scaffold on the intestinal part. Using this device, Lee and Sung showed that butyrate, a compound known to enhance the gut barrier function, attenuated lipid accumulation; a benefit not observed when the drug was tested on hepatocytes alone. Recently, Slaughter et al. (2021) developed a body-on-a-chip with adipose and liver tissue, to evaluate the mechanisms involved in NAFLD development and serve as a drug screening platform. The authors were able to validate the indirect influence of adipocytes on hepatocytes in NAFLD progression. At the cost of greater complexity, the examples above hint that body-on-achip devices may offer a better approximation of the in vivo NAFLD environment.

3D bio-printed liver-like tissues
Three-dimensional bio-printing is another promising technique for the development of in vitro models. 3D structures, including liver-like tissues, are generated via layer-by-layer, spatially controlled deposition of biological materials, biochemicals and cells (van Grunsven, 2017). A comparative analysis of the three most established methods to generate 3D bio-printed liver structures (e.g., inkjet-based, extrusion-based, and photocuring-based bioprinting), is extensively reviewed elsewhere (Murphy and Atala, 2014;Ma et al., 2020). Despite notable advantages, a challenge to the development and widespread use of 3D bio-printed structures is the selection of an appropriate bio-ink. Indeed, the material must show compatibility with both cells and the printing process, while retaining physicochemical -and therefore functional-properties analogous to the organ under consideration. Alginate, gelatin, and collagen are among the most routinely used materials for this purpose. iScience Review Extensive research has been carried out on bio-printed liver structures (Norona et al., 2016;Smith et al., 2004;van Grunsven, 2017). To the best of our knowledge, however, only one 3D bio-printed system has been used to generate the NAFLD phenotype (Kizawa et al., 2017). In this study, a pathological liver tissue was bio-printed using cells harvested from genetically obese Zucker rats with NAFLD. Specifically, the Kenzan method (Moldovan et al., 2017) was used to create a scaffold-free bio-printed structure from liver spheroids composed of rat hepatocytes and mouse fibroblasts. The authors observed high lipid content over 23 days, suggesting that the pathological conditions could be retained and monitored long-term. Despite being designed to investigate drug-induced liver injury, the sophistication of the bio-printed structure proposed by Nguyen et al. (2016) makes it an ideal candidate for the study of NAFLD and NASH. This two-compartment system includes NPC -HUVEC and HSC -seeded at the boundary of each compartment, filled with PHH.
In parallel, due to the shortage of donor organs and inherent risks, researchers are exploring alternative solutions to liver transplant in patients with advanced liver diseases. Emerging approaches in development include liver-like structures bio-printed from HepG2 (Jeon et al., 2017;Ide et al., 2020) or PHH (Kim et al., , 2018 that could pave the way to artificial livers in the future. 3D bio-printing technology may also enhance the assembly of liver-on-a-chip systems, which present challenges such as biological culture preparation (Bhatia and Ingber, 2014). The 3D bio-printed organ-on-achip of Lee and Cho (2016) was devised to overcome limitations such as spatial heterogeneity and the difficulty of providing multiple types of ECM environments when studying cell-ECM interactions (Yeon and Park, 2007). HepG2 cells encapsulated in collagen type I, and HUVEC encapsulated in gelatin were used to construct 2D and 3D hydrogel-based on-chip devices. Similarly, Bhise et al. (2016) developed a liver-on-a-chip with bio-printed HepG2/C3A spheroids. The viability of both models, their extensive lifespan and similarities with intact liver tissue, makes them promising systems for both NAFLD research and drug screening pipelines.
Taken together, although use in NAFLD research is limited, these studies highlight the potential use of 3D bio-printing. However, to enable more widespread use, the costs and specialized knowledge and expertise required to establish and maintain this technology will need to be addressed (van Grunsven, 2017).

USE OF IN VITRO MODELS IN NAFLD DRUG DEVELOPMENT PIPELINES
The majority of clinical trials have focused on patients with NASH and bridging fibrosis, who have an increased risk of developing cirrhosis and adverse outcomes. Depending on the drug mechanism of action, the primary endpoint of interventional trials comprises either NASH resolution without worsening of fibrosis or a minimum of one-stage fibrosis improvement without worsening of NASH. Yet, most clinical trials have been unsuccessful and 4 out of 5 NASH treatments reaching phase III were recently terminated. This suggests monotherapies -individual drugs-might be insufficient to tackle the complex interaction between metabolic, inflammatory and fibrotic pathways that characterize this disease. As a result, evaluation of drug combinations is becoming a major focus of Pharma NASH pipelines. However, despite more than twenty years of intense research activity, no breakthrough has occurred. Indeed, the phase II ATLAS study (NCT03449446) failed to meet the primary endpoint, except for the firsocostat-cilofexor combination, currently subject of further investigation (Gilead Sciences, 2019). While a detailed discussion of the pharmaceutical compounds that have been part of clinical trials does not fall under the scope of this review, we point to extensive recent reviews that address this topic (Fallowfield et al., 2021;Campbell et al., 2021;Guirguis et al., 2021). In order to facilitate the reader's understanding of this section, we have provided two tables that summarize the most relevant information of the ongoing (Table 2) and the terminated/completed (Table 3) phase III clinical trials for pharmaceutical compounds in NAFLD.
In vitro cell culture assays have been used to support pre-clinical development of candidate therapies and to validate the utility of new cellular models. For instance, obeticholic acid (OCA) has been tested in human iPSC-derived hepatocytes (Parafati et al., 2018), a co-culture of human hepatoma cells (Huh7) and HSC (LX-2) (Anfuso et al., 2020) and an organotypic human hepatocyte system (Dash et al., 2017). Boeckmans et al. (2020)    iScience Review determine the efficacy of selonsertib in liver fibrosis (Yoon et al., 2020) or primary rat HSC to evaluate the effects of pioglitazone in vitro (Kawaguchi et al., 2004).
Beyond being suitable models to elucidate NAFLD pathogenesis, liver-on-a-chip systems also play a key role in drug development. To date, Gori et al. (2016), Freag et al. (2021), Cho et al. (2021), Du et al. (2021) and Gori et al. (2021) have used liver-on-a-chip devices to test the ability of polyphenols, OCA, elafibranor, and pirfenidone to ameliorate NAFLD. Interestingly, while the disease-modifying effects of elafibranor were shown in a liver-on-a-chip assay, this did not translate to clinical efficacy in a pivotal phase III NASH trial. As a consequence, the drug program was recently terminated (GENFIT, 2020). These contrasting in vitro versus in vivo observations indicate that preclinical NAFLD models still have a long way to go before reliably simulating human NAFLD and predicting patient responses to therapies.

TRANSLATION OF IN VITRO PLATFORMS
The challenge of identifying an efficacious treatment for the disease is evidenced by the litany of recent clinical trial failures that have encompassed a diverse range of targets. Notwithstanding, many synthetic drugs are currently in development as monotherapy or combination therapy regimens. The availability of reliable human-based in vitro models would help to address this challenge, by both enhancing our understanding of NAFLD progression and predicting the therapeutic response to drug candidates. So far, in vitro models have only managed to recapitulate specific aspects of the in vivo disease phenotype. Indeed, the establishment of multi-dimensional systems that reflect the combined effects of genetics, sex, comorbidities and inter-organ cross-talk remains a pipe dream. Mounting evidence suggests, however, that the complexity of in vitro models will be key to screen drugs and to predict clinically relevant therapeutic responses. This is illustrated in Lee and Sung (2018), where the beneficial effect of butyrate on lipid accumulation only became apparent in a gut-liver-on-chip, but was not detected when the compound was tested using hepatocytes alone.
An ideal NAFLD in vitro model used for drug screening should satisfy biological fidelity and usability requirements. Biological fidelity depends on the selection of appropriate liver cells (parenchymal cells and NPC) and their aggregation in a structure enabling cell-cell and cell-ECM interactions. Future trends will likely see a transition from immortalized cell lines to PSC, due to their almost unlimited supply, life-span and similarity to in vivo human phenotype (Hay et al., 2007;Gao and Liu, 2017). For the observed response to be shaped by inter-organ cross-talk, these systems need to be linked with intestinal, pancreatic and adipose tissue models. 3D liver structures cultured in perfusion devices are quickly developing to meet these standards. Usability of such models relies instead on their lifetime (e.g., a minimum of 2-3 weeks of observation are needed for cytotoxicity testing) and the availability of specialized know-how for their operation.
To ensure clinical relevance, NAFLD in vitro models require validation against known features of in vivo human pathogenic processes or responses to therapeutic agents. Such validation could be performed via an 'omics' approach, which can evaluate the degree of pathological equivalence between an in vitro model and human liver samples at the molecular level, as measured by differentially expressed genes and enriched pathways. Such a strategy has been adopted by Ramli et al. (2020) and Feaver et al. (2016).
Alternatively, models could be assessed by their ability to reproduce results obtained in ongoing clinical trials. For example, Hurrell et al. (2020) demonstrated the potential of their system as a drug screening platform by testing pharmaceuticals that were in phase II or phase III clinical development. However, robust human validation and critical comparative analyses are rarely performed, with most researchers focusing on specific genes and pathways of interest (e.g., the expression level of TGF-a, a-SMA or TNF-b eliciting fibrosis and inflammation) when designing in vitro systems for NAFLD. Ideally, extensively validated platforms would allow more reliable pharmacodynamic-pharmacokinetic and toxicity assays of candidate compounds for NAFLD treatment,  (Dash et al., 2017), have delineated mechanisms of action including modulation of collagen and MMP2 and 9 activity, as well as its possible toxicological effects. Figure 4 shows the different potential applications of in vitro models in translational research for NAFLD.
Despite the advantages they offer, 3D in vitro models remain expensive and inaccessible to new users. Indeed, liver-on-a-chip and bio-printed liver-like tissues are quite complex and steep learning curves are associated with their operation. Ideally, technological advancements and the establishment of rigorous standards will enable these models to become a concrete alternative to animal models in the near future.

FUTURE PERSPECTIVES
Emerging in vitro human-based models have enhanced our understanding of NAFLD evolution and disease mechanisms. However, open challenges remain. Among these, the most prominent are the ability of the model to encompass the whole spectrum of the disease, to reproduce the inter-individual variability observed in NAFLD patients and capture the interaction between multiple organs involved in the pathogenesis of the disease. We expect that in the near future complex in vitro models, such as body-on-achip based on iPSC or PHH, will become the platform of choice to mimic NAFLD establishment and progression. Inclusion of physiologically-based toxicokinetic computer modeling to study ADME (absorption, distribution, metabolism, and excretion) in these systems will likely represent a further step toward the replication of the human liver microenvironment.
The availability of validated preclinical models will streamline the adoption of a quantitative approach to (a) the identification of NAFLD etiology and potential therapeutic targets, and (b) the rational design of pharmacological treatments. These aims are increasingly pursued in a biological systems control perspective, wherein a disease arises due to failure of multi-scale control mechanisms ensuring robustness of biological processes to internal/environmental perturbations. Using this approach, mathematical models and gene regulatory networks reconstructed from 'omics' data provide new insights on the establishment of diseases; and the design of personalized effective treatments can be framed as an engineering control The combination of 'omics' and cell cultures has the potential to enable the study NAFLD patients 'in-the-dish', resulting in the design of effective treatment or identification of predictive and prognostic biomarkers. In vitro NAFLD models, combined with metabolomic and genomic analyses/genetic variant studies, permit the identification of pharmacological targets and susceptible patient subpopulations. Once a putative drug -or combination of drugs -has been indicated as a candidate NAFLD therapy, several pharmacological investigations test the efficacy, cytotoxicity, and possible interactions between compounds. Drugs that are successfully triaged are funneled to clinical evaluation. The ultimate goal -which has not yet been achieved-would employ a precision medicine approach (gray) to the design of patienttailored NAFLD treatments. iScience Review problem. For example, Liao et al. (2020) used a kinetic model of hepatic fructose metabolism to simulate the effect of excessive dietary uptake of this sugar on dyslipidemia observed in early NAFLD patients. The authors identified fructokinase as a molecular target of the fructose pathway and predicted its suppression would revert lipid accumulation. Van Riel et al. (2020) used instead a machine learning algorithm, informed by metabolomic and transcriptomic time-series, to predict the metabolic response induced by treatment with a Liver X Receptor (LXR) agonist. Currently, 3D models are mainly used for biological studies due to their cost and low-throughput. Tackling such limitations will prompt adoption of these in vitro models by the pharmaceutical industry. Indeed, within drug development pipelines, these models would allow only the most promising compounds to funnel toward clinical trials, with obvious economic benefit. If used in combination with disease-relevant ex vivo models (e.g., human precision-cut liver slices) (Palma et al., 2019), in vitro models might help to reduce the (over)reliance on in vivo models for drug efficacy and toxicological studies.
We expect complex in vitro platforms to be increasingly integrated with 'big data' for compound testing, identification of therapeutic targets, and personalized medicine applications. Precision medicine approaches would enable, first, the clustering of NAFLD patients by disease stage and/or genetic variants (e.g., PNPLA3) and the development of in vitro models from patient-derived iPSC on which to assess safety and efficacy of candidate therapies. Second, these studies could identify novel companion biomarkers of NAFLD progression or regression, thereby supporting the design of non-invasive diagnostic tests.
In conclusion, we have provided a comprehensive overview of various in vitro models proposed to study the pathophysiology of NAFLD. The different models available for this disease have specific advantages and disadvantages. Accordingly, the choice of model may depend on the particular context of use.